Cytokines play an important role in regulating the cellular response during inflammation and other immune functions. Of particular interest are the cytokines interleukin-1 (IL-1, .alpha. and .beta.) and tumor necrosis factor (TNF, .alpha. and .beta.) which are the intercellular proteins involved in the initial step of the inflammatory response cascade (Arai, et al., Ann. Rev. Biochem, 59: 783-836 (1990). Thus, there has been a substantial amount of research recently devoted to interfering with the production of IL-1 and TNF in response to an inflammatory stimulus.
One therapeutic approach involves suppressing the production of IL-1 and TNF at the level of transcription and/or translation and/or secretion. The activities associated with certain of pyridinyl imidazoles led to a class of compounds referred to as CSAID.TM. compounds (FIG. 1). These compounds appear to arrest the expression of IL-1 and TNF predominantly at the translational level, although a lesser effect on transcription has also been observed but effects on other steps cannot be ruled out.
The pyridinyl imidazole, 5-(4-pyridyl)-6(4-fluorophenyl)-2,3-dihydroimidazo(2,1-b)thiazole (SK&F 86002) was identified as the prototypic CSAID.TM. compound. The basis for its activity has been established and characterized (Lee, et al., Int'l. J. Immunopharm, 10(7): 835-843 (1988); Agents and Actions 27(3/4): 277-279 (1989) and Int'l. J. Immunother, 6(1):1-12 (1990)). SAR studies suggest that cytokine suppressive effect of the pyridinyl imidazoles represents a unique activity independent of their inhibitory effects on eicosanoid and leukotriene production. However, no compound of the initial series was selective for cytokine suppressive activity or was particularly potent.
Since the CSAID.TM. compounds have substantial potential, inter alia, as novel anti-inflammatory therapeutic agents, there is significant interest in characterizing their mechanism of action at the molecular level, as well as obtaining compounds with increased selectivity and potency. One approach involves the identification and characterization of the molecular targets thereby enhancing the understanding of the biochemical processes involved in inflammation and aid in the design and screening of more potent anti-inflammatory drugs. PCT application WO 95/07922, published 23 March 1995, inter alia, discloses the purification and characterization of one such molecule, a CSAID binding protein (CSBP).
It is now appreciated that the binding of an effector cytokine to its receptor protein is just the first step in a cascade of events. One response to a variety of cellular stimuli, including cytokines, involves a series of protein kinase steps known as the MAP (mitogen-activated protein) kinase cascade. Ammerer, G. (1994) Curr. Opin. Genet. Dev. 4, 90-95; Cobb, et al., (1995) J. Biol. Chem. 270, 14843-14846; and Herskowitz, I., (1995) Cell 80, 187-197. Several genetically distinct MAP kinase pathways have been defined in yeast and at least three exist in mammalian cells.(Ammerer, supra and Cobb et al., supra). The mammalian MAP kinases include the extracellular signal regulated kinases (ERKs) the c-Jun N-terminal kinases (JNKs) and the CSBP/p38/RK/Mpk2 kinases.(Cobb et al., supra). These kinases are activated by distinct upstream dual specificity kinases (MAP kinase kinases) which phosphorylate both threonine and tyrosine in a regulatory TXY (Thr-Xaa-Tyr, where X is any amino acid) loop present in all MAP kinases.(Hanks et al., (1988) Science 241, 42-52.). Once activated, these MAP kinases phosphorylate their substrates on serine and/or threonine residues with attendant effects on their activity. For example, phosphorylation of c-Jun and ATF2 by JNK (Gupta, et al., (1995) Science 267, 389-393; and Derijard, et al., (1994) Cell 76, 1025-1037) stimulates their transcriptional activity.
CSBP (also known as p38, RK and mpk2) (Lee, et al., (1994) Nature 372, 739-746; Han, et al., (1994) Science 265, 80-881; and Rouse et al., (1994) Cell 78, 1027-1037) is the mammalian homologue of the yeast Hog1 protein which is required for growth of yeast in high osmolarity media (Brewster, et al., (1993) Science 259, 1760-1762) and it can partially complement a hogl deficiency in yeast.(Han et al., supra; and Kumar, et al. (1995) J. Biol. Chem. 270, 29043-29046). CSBP is activated in mammalian cells by environmental or chemical stress such as hyperosmolarity, UV light, heat shock, arsenite, and endotoxin or cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) (Lee, et al., supra; Han, et al., supra; Rouse et al, supra; Freshney, et al., (1994) Cell 78, 1039-1049; and Raingeaud, et al., (1995) J. Biol. Chem. 270, 7420-7426). In response to stress, CSBP kinase activity is activated through phosphorylation by at least two MAP kinase kinases, MKK3 and MKK4 (also known as SAPK) (Derijard, et al., (1995) Science 267, 682-685; and Lin et al., (1995). Science 268, 286-290). Of the in vitro substrates of CSBP which include MAPKAP kinase-2.(Rouse et al., supra; Freshney et al., supra; and Cuenda, et al., (1995) FEBS Lett. 364, 229-231), myelin basic protein (MBP) (Lee et al., supra; and Kumar et al., supra), and ATF2 (Derijard, et al., supra), only MAPKAP kinase-2 is known to be an in vivo substrate, since pretreatment of cells with 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole, a specific inhibitor of CSBP, blocks the activation of MAPKAP kinase-2. In turn, MAPKAP kinase-2 phosphorylates the small heat shock proteins HSP25/27 in vitro and in vivo (Rouse et al., supra; Freshney et al., supra and Cuenda et al., supra). Inhibitors of CSBP also block the production of inflammatory cytokines from LPS stimulated human monocytes (Lee et al., supra) and IL-1 stimulated endothelial cells (Lee, et al.,, (1993) Ann N. Y. Acad. Sci. 696, 149-170), and more recently CSBP has been implicated in the apoptosis of neurons upon growth factor removal (Xia, et al., (1995) Science 270, 1326-1331).
Given the many signals which activate CSBP, and its potential involvement in several cellular responses, it is of interest to discover further activators and substrates. As disclosed herein, human CSBP was used as the bait in a yeast two-hybrid screen.(Fields, et al., (1989) Nature 340, 245-247) to identify a serine-threonine protein kinase, MAPKAP kinase-3, which binds to and is an in vivo and in vitro substrate of CSBP. This kinase was disclosed by Sithanandam, G. et al., in "Tyrosine Phosphorylation and Cell Signaling" (Abst) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., May 3-7, 1995, page 172 and in Mol. Cellular Biol, 16(3): 868-76 (1996).
The DNAs of this invention, such as the specific sequences disclosed herein, are useful in that they encode the genetic information required for the expression of the novel MAPKAP kinases. Additionally, the sequences may be used as probes in order to isolate and identify any additional members of the MAPKAP kinase family as well as forming the basis of antisense therapy for disease conditions which are characterized by atypical expression or activation of the MAPKAP kinase genes. The novel protein itself is useful directly as a diagnostic agent as well as a component in a screening system for compounds which are antagonists or agonists of effector activity. The protein is also useful for eliciting antibody production in heterologous species, said antibodies being useful for the aforesaid diagnostic, therapeutic and screening applications. These and additional uses for the reagents described herein will become apparent to those of ordinary skill in the art upon reading this specification.